Transferrins synergistic anions

Fig. 22. A selection of synergistic anions bound by transferrins. From Schlabach and Bates (178).

Fig. 23. Visible absorption spectra for diferric transferrin complexes utilizing various synergistic anions, showing the variation in mal for the charge transfer band. Anions are 1, nitrilotriacetate 2, carbonate 3, salicylate 4, thioglycolate 5, glycine 6, glyoxy-late and 7, glycolate. From Schlabach and Bates (178), with permission.

Fig. 26. Binding modes for anions other than carbonate. In (a) the mode of binding of oxalate to human lactoferrin, as determined crystallographically (192,193), is shown. In b is a generalized model for synergistic anion binding to transferrins, based on EPR studies (191) and the crystallographic results for oxalate. From Shongwe et al. (192), with permission.

Spectroscopic studies have consistently demonstrated the existence of multiple conformational states for the metal sites in transferrins, especially when using metal ions other than Fe3+ and anions other than C032. The differences are not necessarily related to intrinsic geometrical differences between the two sites in each molecule, but also reflect changes dependent on pH, the nature of the synergistic anion, or salt effects. 

Distinct differences are also seen when anions other than C032 are used. The crystal structure of oxalate-substituted diferric lactoferrin shows differences in the anion binding in the two sites in the C-site the oxalate is symmetric bidentate, whereas in the N-site it is asymmetric (193). When Cu2+ is the metal ion the oxalate binding differences become even more pronounced. Copper-transferrin binds oxalate only in its N-terminal site (91). Copper-lactoferrin and copper-ovotransfer-rin each bind two oxalate ions but binding occurs preferentially in the C-lobe (157,192). These different affinities mean that hybrid complexes can be prepared with oxalate in one site and carbonate in the other (92, 157, 192). The use of oxalate as synergistic anion gives rise to spectroscopically distinct sites for other metal ions also (171). 

The above mechanism is totally consistent with the crystallographic results from the various forms of lactoferrin and transferrin (Section III.B). These lead to a structural model of binding shown pictorially in Fig. 29. In the first step the synergistic anion (usually carbonate) is bound in the specific site on domain 2 of each lobe. Binding may be preceded by electrostatic attraction from the exposed helix N-termini and several basic sidechains in the open interdomain cleft. 

Fligh-spin iron in a nonheme environment exhibits a significant change in the thermodynamics and kinetics of protein binding on reduction from Fe " to Fe ". This is illustrated by the mammalian serum iron-transport protein, transferrin. The thermodynamic affinity for Fe " is 10 in the presence of carbonate as a synergistic anion, and is reduced to 10 on reduction to Pg2-i- 21,22 jj-on-ligand turnover is also enhanced upon reduction. The net result is a non-Nernstian spectroelectrochemical response because of an elec-trochemically driven reduction followed by a coupled equilibrium dissociation of Fe " as illustrated in eqns (2.4) and (2.5) ... 

The storage of iron in humans and other mammals has been dealt with in the previous section. Only a small fraction of the body s inventory of iron is in transit at any moment. The transport of iron from storage sites in cellular ferritin or hemosiderin occurs via the serum-transport protein transferrin. The transferrins are a class of proteins that are bilobal, with each lobe reversibly (and essentially independently) binding ferric ion. This complexation of the metal cation occurs via prior complexation of a synergistic anion that in vivo is bicarbonate (or carbonate). Serum transferrin is a monomeric glycoprotein of molecular weight 80 kDa. The crystal structure of the related protein, lactoferrin, has been reported, and recently the structure of a mammalian transferrin" has been deduced. 

Biochemical experiments and crystal structures of apo, mono, and diferric serum transferrin greatly enhance our understanding of the mechanism by which Tfs strongly chelate ferric ion and then release it within the endosome. These processes are inherently related the iron-binding steps occur in the opposite order to the steps leading to iron release from transferrin. Binding of iron to the tyrosine residues and to the synergistic anion while the Tf lobe is in the open conformation appears to be the first step. " " Once the ferric-loaded domain samples the closed... 

UV-difference spectroscopy, and other studies in vitro with plutonium (IV), tho-rium(IV) and a number of trivalent lanthanides, have demonstrated that, like iron, two lanthanide or actinide metal atoms are bound per transferrin molecule (Duffield and Taylor 1986, Taylor et al. 1991, Zak and Aisen 1988). Saturation of the lanthanide- or actinide-containing transferrin with excess iron results in a liberation of the f element, thus suggesting that these metals are binding to the two iron-binding sites on the transferrin molecule (Taylor et al. 1991). These studies have also shown that bicarbonate is necessary, as a synergistic anion, for the binding of actinides and lanthanides to transferrin. 

---